The stability has to be maintained in a substance to keep the substance normally functioning. In particular, the stability is a key factor determining shelf life and half life in vivo especially in a substance composed of proteins that are easily denaturated. Thus, to develop a therapeutic agent or diagnostic reagent comprising proteins, it is necessary to improve the stability of candidate compositions for those agents. Protein is weak in keeping its natural form and thus easily inactivated and changed in structure by heat or a chemical denaturant. In the case of the serine based protease inhibitor including antitrypsin, antichymotrypsin and alpha-1-antitrypsin, a polymer is formed in between molecules by heat treatment, resulting in the inactive form.
The conventional therapeutic agent comprising proteins separated and purified from human blood might have problems of limitation of supply and potential contamination with infectious materials such as AIDS or hepatitis virus. Therefore, other attempts have been made to produce a protein therapeutic agent by genetic engineering techniques. However, the recombinant proteins prepared by genetic engineering technique have lower stability than the conventional protein therapeutic agent comprising the proteins separated from human blood. Besides, when the recombinant protein is administered into blood plasma, half-life of the protein decreases rapidly, putting the usability of such recombinant protein as a therapeutic agent in doubt. Attempts have been made to overcome the problem of the lower stability. One of the attempts is the genetic engineering method to produce a mutated protein with improved stability without changing its activity by substituting amino acids via gene mutation. Heat-resistance of a protein is closely associated with stability against degeneration of a protein (Pace, Trends in Biotechnology, 8, 93-98, 1990).
AT is synthesized in liver cells and then secreted into blood. Along with most serine based protease inhibitors existing in blood plasma including trypsin, chymotrypsin, elastase, collagenase, thrombin and plasmin, AT belongs to serpin family. AT is a glycoprotein of 52 kD in molecular weight and is physiologically functioning as an inhibitor to elastase of neutrophiles. In particular, AT prevents elastic fiber from being decomposed by elastase of neutrophiles.
Many cases of innate genetic mutation that causes AT-related pathological symptoms have been reported (Carrell et al., Mol. Biol. Med. 6, 35-42, 1982). In most cases, the decrease of AT level in blood plasma breaks the balance between protease and its inhibitor and therefore the lungs lose flexibility, which develops into emphysema (Gadek and Crystal, in Metabolic Basis of Inherited Disease. Stanbury et al., Eds., McGraw-Hill, New York. pp. 1450-1467).
In addition to the emphysema caused by genetic defection, heavy smoking or serious environmental pollution causes inactivation of protease inhibitor to cause emphysema. To overcome this disease largely found in Caucasian of Northern America and Europe, AT market of at least 100 million dollars/year has been formed and AT extracted from blood has been actually administered as a therapeutic agent.
AT can also be used for the treatment of acute shock syndrome (Robin W. Carrell, Biotechnology and Genetic Engineering Reviews. 4, 291-297 (1986)). Shock syndrome is caused by unbalance between sulfin and protease in blood plasma resulted from the sudden mass-release of neutrophiles. Considering limitation of obtaining the raw material and possible virus infection, preparation methods by genetic engineering techniques have been tried as an alternative.
The nucleotide sequence of DNA encoding AT protein has been already identified (Long et al., Biochemistry 23, 4828 (1984)) and AT gene was expressed in E. coli (Bollen et al., FEBS Lett. 16, 67 (1984); Courtney et al., Proc. Natl. Acad. Sci. USA 81. 669 (1984); Tessier et al., FEBS Lett. 208, 183 (1986); Johnsen et al., Mol. Biol. Med. 4, 291 (1987); Sutiphong et al., Mol. Biol. Med. 4, 307 (1987); Lee and Yu, Journal of Biochemistry and Molecular Biology 22, 148 (1989); Lee et al., Molecules and Cells 3, 71-74 (1993)) or yeast (Travis et al., J. Biol. Chem. 260. 4384 (1985); Rosenberg et al., Nature 312, 77 (1984); Cabezon et al., Proc. Natl. Acad. Sci. USA 81, 6594 (1984); Kim et al., Journal of Biochemistry and Molecular Biology 23, 236 (1990); Kim et al., Korean Journal of Microbiology 30, 108 (1992)) according to previous reports.
There was a successful attempt, in which AT activation site, the 358th methionine residue was substituted with another amino acid residue by site specific mutagenesis to develop an inhibitor to serine based protease other than elastase or to develop an inhibitor with improved resistance against oxidation (Rosenberg et al., Nature 312, 77-80 (1984); Courtney et al., Nature 313, 149-151 (1985); Barr et al., U.S. Pat. No. 4,732,973; Insley et al., U.S. Pat. No. 4,711,848). The non-glycosylated AT produced in yeast exhibited reduced heat-resistance in vitro and the decrease of heat-resistance was allegedly closely related to the decrease of its half-life in vivo (Travis et al., J. Biol. Chem., 260. 4384 (1985)).
The co-relation between AT structure and AT functions was explained well by Huber and Carrell (Biochemistry 28, 8951-8963 (1989)).
The present inventors continued to study to overcome the problem of instability of AT prepared by genetic engineering techniques. As a result, the present inventors completed this invention by confirming that the 168th and the 189th amino acid residues of AT were substituted with cysteines and the protein was oxidized, resulting in recombinant mutein AT with a disulfide bond, which had much more improved heat-resistance and thermodynamic stability, compared with the wild type recombinant AT or the patent granted F51L/M374I mutein (Korean Patent No. 133252).